Effect Of Chronic Morphine Exposure Research Articles

Chronic Morphine Induces Adaptations in Opioid Receptor Signaling in a Thalamostriatal Circuit That Are Location Dependent, Sex Specific, and Regulated by μ-Opioid Receptor Phosphorylation.

Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) via activity at μ-opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in the DMS. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation) in male, but not female, mice. At MThal cell bodies, chronic morphine treatment decreased subsequent morphine activation of potassium conductance (morphine tolerance) in both male and female mice. In knock-in mice expressing phosphorylation-deficient MORs, chronic morphine treatment resulted in tolerance to, rather than facilitation of, subsequent morphine signaling at MThal-DMS terminals, suggesting phosphorylation deficiency unmasks adaptations that counter the facilitation observed at presynaptic terminals in wild-type mice. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry, and sex.

Chronic Exposure to Morphine Leads to a Reduced Affective Pain Response in the Presence of Hyperalgesia in an Animal Model of Empathy.

BackgroundEmpathy is the capability to represent the mental and emotional states of other subjects. Previous studies have demonstrated a possible correlation between morphine addiction and altered empathy response in morphine-addicted subjects. This study was performed to evaluate the effect of chronic morphine exposure as an animal model of morphine addiction on empathic changes in affective and sensory pain.MethodsAdult male Wistar rats (3 months old) were used for the current study. Animals were grouped in cages of two (n = 8 for each group) and one animal was selected as the pain observer group. Pain observer animals received either saline or morphine (10 mg/kg, twice daily for 8 days). At ninth day, formalin [50 µg, 5%, subcutaneous (SC)] was injected into the hindpaw of the cagemate and placed inside the cage. Elevated plus maze (EPM) and open field test (OFT) were recruited to evaluate anxiety; hot plate and tail flick tests were used to assay sensory pain. Conditioned place aversion (CPA) was also measured as indicator of affective pain component.FindingsChronic morphine exposure led to a reduced level of anxiety in EPM and OFT assays. An opioid-induced hyperalgesia was observed in the sensory pain assays, while there was a reduced affective pain in the CPA paradigm in morphine-treated animals.ConclusionIt might be plausible that chronic morphine exposure might alter empathy for pain through affective and not sensory pain pathways.

A54 DELTA OPIOID RECEPTOR SIGNALING MEDIATES OPIOID INDUCED TOLERANCE AND HYPERALGESIA IN COLONIC SENSORY NERVES

Abstract Background While opioid drugs are widely used for treating abdominal pain, prolonged exposure to opioids can induce tolerance and paradoxically increase pain. We previously showed in colonic afferent nerves that chronic morphine treatment causes tolerance and paradoxical hyperalgesia, however the mechanisms are unknown. Aims To evaluate the role of delta opioid receptor (DOR) signaling in opioid induced tolerance and hyperalgesia in colonic nerves during chronic morphine exposure. Methods C57BL/6 mice were injected (i.p.) twice daily for 7 days with escalating doses of morphine (10, 20, 30 mg/kg for the first 3 days, then 40mg/kg on 4th to 7th day); a subset of mice were also injected with 2.5 mg/kg naltrindole (NTI), a DOR antagonist. The analgesic response in vivo was monitored daily using the tail-flick test. To assess the effect of chronic morphine exposure, mice were euthanized on day 8, and the dorsal root ganglia (DRGs) and colons were harvested. Isolated DRG neurons were incubated with 1μM morphine (30 min) and neuronal excitability was determined by measuring the rheobase (amount of current required to elicit an action potential) using perforated patch clamp. To assess changes in colonic afferent nerve excitability, ex vivo afferent nerve recordings were obtained from flat-sheet colon preparations to determine the effects of 1μM morphine (10 min perfusion) on mechanosensitivity by probing with 1g von Frey hair. Results The tail flick test showed that the DOR antagonist NTI inhibited (30%) the maximal antinociceptive effect of morphine and reduced morphine tolerance. In patch clamp recordings, 1μM morphine paradoxically increased the excitability of small DRG neurons from morphine mice (rheobase decreased 28%; p<0.05, 2-way ANOVA), whereas this excitatory effect was absent on neurons from NTI+morphine mice. Acute application of 1μM morphine had no effect on afferent nerve responses to probing in morphine mice (14.9 vs 16.7 Hz; p=ns, paired t test, n=15) whereas it inhibited the afferent nerve response to probing in NTI+morphine mice (15.2 vs 9.9 Hz; p<0.001, paired t test, n=16). Furthermore, most single units from morphine mice had an increased response to probing following acute morphine application (7/15 vs 2/16 units excited), whereas most units form NTI-morphine mice were inhibited (14/16 vs. 3/15 units inhibited, P<0.001, Chi-square). Conclusions These findings suggest that DOR signaling plays a significant role in the development of opioid tolerance and hyperalgesia in colonic afferent nerves induced by chronic morphine treatment. Thus, antagonists of DOR may help to mitigate these side effects induced by opioids. Funding Agencies CCC

Alterations in brain activation in response to prolonged morphine withdrawal-induced behavioral inflexibility in rats.

The inability to stop a repetitive maladaptive behavior is a main problem in addictive disorders. Neuroadaptations that are associated with behavioral inflexibility may be involved in compulsive drug use. The aim of the present study was to investigate the pattern of behavioral inflexibility during morphine withdrawal and map brain activation that is linked to alterations in flexibility. We first analyzed the effects of chronic morphine exposure on reversal learning after 2-week (short-term) and 6-week (prolonged) morphine withdrawal. We then compared the level of neuronal activation using cFos immunohistochemistry in 15 brain areas between rats that underwent morphine withdrawal and saline-control rats after a test of reversal learning. Only prolonged morphine withdrawal impaired reversal learning. Rats that exhibited impairments in reversal learning presented a significant decrease in cFos expression in the orbitofrontal cortex (OFC), including the medial, lateral, and ventral OFC. cFos expression significantly increased in the dorsomedial striatum and major subregions of the medial prefrontal cortex (mPFC) in the morphine group. Rats that underwent prolonged morphine withdrawal exhibited no significant changes in cFos expression in the dorsolateral striatum, nucleus accumbens, amygdala, paraventricular thalamic nucleus, or motor cortex. The rats that underwent short-term withdrawal did not present any changes in cFos expression in any of these brain regions. Altogether, these data suggest that alterations in the function of the frontal cortex and its striatal connections during the late morphine withdrawal phase may underlie the disruption of inhibitory control in opioid dependence.

Chronic Morphine Increases Fos‐Positive Neurons After Concurrent Cornea and Tail Stimulation

The aim of the present study was to examine the effect of chronic morphine exposure on diffuse noxious inhibitory controls in a large population of neurons throughout the medullary dorsal horn, as assessed using immunocytochemistry for c-Fos protein. Overuse of medications, including the opioids, to treat migraine headache can lead to progressively more frequent headaches. In addition, chronic daily headache sufferers and chronic opioid users both lack the inhibition of pain produced by noxious stimulation of a distal body region, often referred to as diffuse noxious inhibitory controls. In urethane anesthetized rats, Fos-positive neurons were quantified in chronic morphine and vehicle-treated animals following 52°C noxious thermal stimulation of the cornea with and without the application of a spatially remote noxious stimulus (placement of the tail in 55°C water). When compared to chronic morphine-treated animals that did not receive the spatially remote noxious stimulus, chronic morphine-treated animals given corneal stimulation along with the spatially remote noxious stimulus demonstrated a 163% increase (P < .05) in the number of Fos-positive neurons in the superficial laminae of the medullary dorsal horn and a 682% increase (P < .01) in deep laminae that was restricted to the side ipsilateral to the applied stimulus. In contrast, no significant difference was found in Fos-like immunoreactivity in vehicle-treated animals given concurrent cornea and tail stimulation or only cornea stimulation in either superficial or deep laminae. It is proposed that an increase in descending facilitation and subsequent loss of diffuse noxious inhibitory controls contributes to the development of medication overuse headache.

Differential Changes in Expression of Metabolic-related Neuropeptides in the Rat Brain during Morphine Withdrawal-induced Anorexia

Event Abstract Back to Event Differential Changes in Expression of Metabolic-related Neuropeptides in the Rat Brain during Morphine Withdrawal-induced Anorexia B. Pintér-Kübler1, S. Ferenczi1, A. Molnár1, C. Núñez2, Z. Szőke1, M. V. Milanés2 and K. J. Kovács1* 1 Institute of Experimental Medicine, Hungary 2 University of Murcia, Spain In this study we followed the effects of chronic morphine exposure and naloxone-precipitated morphine withdrawal on energy balance, metabolic related hormone secretion as well as on the transcription of anorexigenic and orexigenic neuropeptides within the hypothalamus. Animals were implanted with morphine pellets and on day 8, morphine withdrawal was precipitated by an injection of naloxone. Rats were decapitated at various time intervals after injection. Food consumption and the body weight change were recorded daily. Morphine treated animals consumed significantly less food than placebo implanted controls and it was accompanied by a reduction in body weight gain. Morphine withdrawal resulted in elevation of CRH and UCNII expression and a decline in POMC levels. The relative quantity of POMC and CART mRNAs in the hypothalamic samples gradually decrease, while NPY expression is only transiently upregulated. Morphine withdrawal results in serious affective and somatic symptoms including activation of the hypothalamo-pituitary-adrenocortical (HPA) axis. The plasma levels of ACTH were increased and lasted up to 2 hours after administration of naloxone.The present data suggest a possible role of hypothalamic CRH and UCNII,- rather than POMC/CART system in mediation of metabolic changes such as weight loss during naloxone precipitated morphine withdrawal in rats. Keywords: Behaviour and cognition, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Behaviour and cognition Citation: Pintér-Kübler B, Ferenczi S, Molnár A, Núñez C, Szőke Z, Milanés MV and Kovács KJ (2011). Differential Changes in Expression of Metabolic-related Neuropeptides in the Rat Brain during Morphine Withdrawal-induced Anorexia. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00021 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. K. J Kovács, Institute of Experimental Medicine, Budapest, Hungary, kovacs@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers B. Pintér-Kübler S. Ferenczi A. Molnár C. Núñez Z. Szőke M. V Milanés K. J Kovács Google B. Pintér-Kübler S. Ferenczi A. Molnár C. Núñez Z. Szőke M. V Milanés K. J Kovács Google Scholar B. Pintér-Kübler S. Ferenczi A. Molnár C. Núñez Z. Szőke M. V Milanés K. J Kovács PubMed B. Pintér-Kübler S. Ferenczi A. Molnár C. Núñez Z. Szőke M. V Milanés K. J Kovács Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Theta pulse stimulation: A natural stimulus pattern can trigger long-term depression but fails to reverse long-term potentiation in morphine withdrawn hippocampus area CA1

The effects of chronic morphine exposure on synaptic plasticity in the CA1 region of the hippocampal slice preparation using extracellular recordings of the population spike (PS) evoked in response to Schaffer collateral stimulation were studied. High frequency stimulation (HFS; 1X100 Hz) and theta pulse stimulation (TPS; 5 Hz trains for 3 min) were used as patterned activities. The results showed that in rats chronically treated with morphine (dependent group), TPS induced long-term depression (LTD) of PS in CA1 in the absence of in vitro morphine. This TPS-induced PS LTD was blocked in the presence of either AP5 (NMDAR antagonist) or CPX (A1 adenosine receptor antagonist) alone, but was not blocked when AP5 and CPX were co-applied. This TPS-induced PS LTD was also blocked in the presence of either 8-PT (a selective A1 adenosine receptor antagonist) or MRS1220 (a specific A3 receptor antagonist). Additionally, when TPS was applied prior to HFS, PS long-term potentiation (LTP) was blocked. However, when TPS was applied after HFS, there was no reversal of PS LTP in slices from dependent rats in contrast to controls which displayed reversal of LTP. Both the PS LTD and the absence of PS LTP reversal were blocked by in vitro application of morphine. It is concluded that morphine withdrawal was associated with greater depression of CA1 PS elicited by natural stimulus induced activity pattern. This effect was associated with changes in NMDA and adenosine receptors due to chronic morphine administration. Such an in vitro preparation could provide a novel paradigm to investigate withdrawal effects on synaptic plasticity.

Pre‐ and postsynaptic regulation of locus coeruleus neurons after chronic morphine treatment: a study of GIRK‐knockout mice

While the acute inhibitory effect of opioids on locus coeruleus (LC) neurons is mediated primarily by the activation of G protein-gated inwardly-rectifying K(+) (GIRK) channels, the 3'-5'-cyclic adenosine monophosphate (cAMP) system has been implicated in the effects of chronic morphine exposure. Presently, the impact of chronic morphine treatment on GIRK-dependent and GIRK-independent mechanisms underlying the opioid-induced inhibition of LC neurons is unclear. Here, opioid-induced postsynaptic inhibition was studied in LC neurons from wild-type and GIRK2/GIRK3(-/-) mice at baseline and following chronic morphine treatment. The postsynaptic inhibition of LC neurons caused by the opioid agonist [Met](5) enkephalin (ME) was unaffected by chronic morphine treatment in mice of either genotype. Furthermore, chronic morphine treatment had no effect on the forskolin augmentation of the ME-induced current in wild-type LC neurons and only a minor effect on the ME-induced current in LC neurons from GIRK2/GIRK3(-/-) mice. Chronic morphine treatment did, however, lead to an increased frequency of spontaneous excitatory postsynaptic currents (EPSCs) in the LC. Interestingly, while forskolin augmented the EPSC frequency similarly in untreated and morphine-treated wild-type mice, as well as untreated GIRK2/GIRK3(-/-) mice, it failed to increase the frequency of EPSCs in morphine-treated GIRK2/GIRK3(-/-) mice. Altogether, the findings suggest that chronic morphine treatment exerts little impact on ion channels and signaling pathways that mediate the postsynaptic inhibitory effects of opioids but does enhance excitatory neurotransmission in the mouse LC.

Effect of chronic morphine exposure on response properties of rat barrel cortex neurons

Chronic exposure to morphine can impair performance in tasks which need sensory processing. Using single unit recordings we investigate the effect of chronic morphine exposure on the firing properties of neurons in layers IV and V of the whisker-related area of rat primary somatosensory cortex. In urethane-anesthetized animals, neuronal activity was recorded in response to principal and adjacent whisker deflections either stimulated independently or in a conditioning test paradigm. A condition test ratio (CTR) was calculated for assessing the inhibitory receptive field. In layer IV, chronic morphine treatment did not change the spontaneous discharge activity. On responses to principal and adjacent whisker deflections did not show any significant changes following chronic morphine exposure. The magnitude Off responses to adjacent whisker deflection decreased while its response latency increased. In addition, there was a significant increase in the latency of Off responses to principal whisker deflection. CTR did not change significantly following morphine exposure. Layer V neurons, on the other hand, did not show any significant changes in their spontaneous activity or their evoked responses following morphine exposure. Our results suggest that chronic morphine exposure has a subtle modulatory effect on response properties of neurons in barrel cortex.

Effect of long-term exposure of SH-SY5Y cells to morphine: a whole cell proteomic analysis

BackgroundOpiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure.ResultsThus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the μ-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses.ConclusionA total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling.

DNA vaccine-encoded glycoprotein B of HSV-1 fails to protect chronic morphine-treated mice against HSV-1 challenge

The use of morphine has been demonstrated to increase susceptibility to infections. Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen among immunocompromised individuals. In the present study, due to the importance of HSV vaccination in morphine abusers, the effects of chronic morphine exposure on the host response to a HSV-1 gB DNA-based vaccine have been investigated. The study is addressing an important aspect of vaccine development among the susceptible (immunocompromised) hosts. BALB/c mice were exposed to morphine over 11 days. They were then vaccinated with DNA vaccine or KOS strain as a live vaccine. The findings showed that the morphine-treated animals failed to respond to DNA vaccination evaluated by the anti-HSV gB antibody titer, delayed type hypersensitivity (DTH) and lethal HSV-1 challenge. Under the same conditions, the KOS vaccine showed a reduced Ab titer and DTH response in morphine-treated mice, but could protect mice against the lethal challenge and was safe for vaccination of morphine-treated animals.

Chronic morphine exposure impairs short-term synaptic depression of geniculo-cortical visual pathway in vivo

Chronic morphine exposure can induce addiction and affect synaptic plasticity, but the underlying neuronal mechanisms remain unknown. Two forms of short-term synaptic depression (paired-pulse depression (PPD) and frequency depression) were investigated in vivo in the geniculo-cortical visual pathway of morphine-treated and saline-treated (as control) adult rats. Acute exposure to morphine had no effect on paired-pulse synaptic depression and 10–40 Hz induced frequency synaptic depression. However, chronic morphine exposure reduced markedly the paired-pulse depression and frequency depression at 40 Hz. The effect of chronic morphine exposure on short-term synaptic plasticity in the geniculo-cortical visual pathway was sensitization given that morphine re-exposure further significantly reduced the short-term synaptic depression. Interestingly, the further reduction in short-term synaptic depression due to re-exposure of morphine was recovered to normal (control) levels at 3 to 6 h after morphine re-exposure. These findings suggest that chronic morphine treatment could significantly degrade the short-term synaptic plasticity of geniculo-cortical visual pathway.

Chronic morphine exposure induces degradation of receptive field properties of LGN cells in cats1

To investigate the effect of chronic morphine exposure on the receptive field properties of lateral geniculate nucleus (LGN) neurons in cats. Cats were injected with morphine (10 mg/kg) or saline twice daily, for 10 d. Subsequently, extracellular single-unit recording techniques were used to examine the sensitivity of LGN neurons to visual stimuli in chronic morphine-treated and saline-treated cats. Compared with saline-treated cats (as controls), LGN neurons in morphine-treated cats had decreased signal-to-noise ratios (1.9 vs 3.1), and orientation and direction sensitivity (0.103 vs 0.135 and 0.074 vs 0.10, respectively), accompanied by significant increases in spontaneous (27.4 vs 17.5) and evoked activity (preferred: 42.2 vs 38.1; average: 28.1 vs 22.3). Chronic morphine exposure can lead to the functional degradation of LGN neurons in cats, which might result from the effects of chronic morphine exposure on inhibitory neurotransmission.

Degradation of response modulation of visual cortical cells in cats with chronic exposure to morphine

The primary visual cortex (V1) plays an important role in vision and visual perception. Studies in many brain regions demonstrate that opiate abuse can change excitatory and inhibitory neurotransmission. To investigate the effect of chronic morphine exposure on the response modulation of V1 simple and complex neurons, we carried out in vivo extracellular recordings in V1 of morphine- and saline-treated (control) cats. Response modulation was quantified as the ratio of first Fourier components (F1) to the mean response (F0). Compared with saline-treated cats, V1 neurons in morphine-treated cats exhibited weaker response modulation and a longer time course of response. The decrease of response modulation was caused by an increase of F0. Further, morphine re-exposure significantly improved the response properties of V1 neurons in morphine-treated cats. These results suggest that chronic morphine treatment leads to a significant degradation of response modulation of V1 neurons and a morphine dependence of primary visual cortical function.

Effect of chronic morphine exposure on the synaptic plasma‐membrane subproteome of rats: a quantitative protein profiling study based on isotope‐coded affinity tags and liquid chromatography/mass spectrometry

The effect of chronic morphine exposure on the synaptic plasma-membrane subproteome in rats was studied by the isotope-coded affinity tag (ICAT) method coupled with capillary reversed-phase liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry. ICAT-labeled tryptic peptides of synaptic membrane proteins were successfully identified using tandem mass spectrometry in conjunction with protein database searching. Several important synaptic plasma-membrane proteins displayed significant regulation changes as a result of chronic morphine exposure in vivo. In particular, an integral membrane protein Na(+)/K+ ATPase (alpha-subunit) involved in regulation of the cell membrane potential by controlling sodium and potassium ion permeability was downregulated by 39 +/- 2%. This result was in excellent agreement with the reduction in electrogenic Na+, K+ pumping due to about 40% downregulation of Na(+)/K+ ATPase alpha3-isoform in myenteric S-neurons of morphine-exposed guinea-pigs measured by others via immunohistochemistry. The decrease in the abundance of non-erythroid alpha II-spectrin in the synaptic plasma-membrane fraction was also observed, which was hypothetically associated with the breakdown of the protein due to the upregulation of the proteolytic enzyme caspase-3 upon chronic morphine exposure.

Basic Science: Mechanisms of Medication Overuse Headache

Headache caused by overuse of symptomatic medications to treat headache represents a challenging clinical problem. Recent studies exploring both the mechanisms of migraine headache and neuroplastic changes following sustained exposure to analgesic drugs suggest insights regarding the pathophysiology of medication overuse headache. In this review, changes that occur following chronic morphine that may be particularly relevant to the induction of medication overuse headache will be discussed. Peripherally, these changes include increased expression of calcitonin gene‐related peptide (CGRP) in primary afferent neurons. Centrally, they include increased descending facilitation from the rostral ventromedial medulla and increased excitatory neurotransmission at the level of the dorsal horn. Interestingly, many of these same changes, including increased CGRP levels and peripheral and central sensitization, are apparent in inflammatory pain states. Recent revelations into the mechanisms of migraine headache suggest that neurogenic inflammation leads to both peripheral and central sensitization in the migraine sufferer. Furthermore, CGRP plays a prominent role in initiating vasodilation of the intracranial blood vessels and subsequent headache. Given the many parallels between the effects of chronic morphine exposure and processes that occur during migraine, it is evident that overuse of symptomatic medications to treat headache could lead to worsening of symptoms. In animals, the neural adaptations that occur after sustained morphine exposure are manifested behaviorally as an increased sensitivity to both tactile and thermal stimulation. In the headache sufferer, these adaptations could lead to medication overuse headache.

Chronic Morphine Treatment Inhibits Opioid Receptor Desensitization and Internalization

Chronic opioid receptor (OR) activation by morphine causes distinct cellular adaptations responsible for the development of tolerance. The present study examines the effect of chronic morphine exposure on the ability of high-efficacy agonists to mediate delta-OR (DOR) and mu-OR (MOR) uncoupling and internalization, two regulatory mechanisms contributing to rapid desensitization of OR function. Chronic morphine treatment (1 microm; 72 hr) of DOR carrying neuroblastoma x glioma (NG108-15) hybrid cells, a prototypical model system frequently used to study cellular aspects of opioid tolerance, completely blocked the capacity of [d-Ala2, d-Leu5]enkephalin (DADLE) and etorphine to desensitize opioid-stimulated [35S]GTPgammaS binding and to mediate DOR internalization. Similar findings were obtained on stably DOR- and MOR-transfected human embryonic kidney (HEK) 293 cells. Chronic morphine treatment also heterologously impaired agonist regulation of non-opioid G-protein-coupled receptors, such as the m(4)-muscarinic acetylcholine receptor and the brain-type cannabinoid receptor. As a possible underlying mechanism, we found that chronic morphine treatment completely blocked agonist-induced redistribution of beta-arrestin1 in both NG108-15 and stably MOR-transfected HEK293 cells. Moreover, attenuation of beta-arrestin1 function appears to depend on persistent stimulation of MAP kinase activity during the course of chronic morphine treatment, because coincubation of the cells together with the MAP kinase blocker PD98059 fully restored beta-arrestin1 translocation and receptor internalization. These results demonstrate that chronic morphine treatment produces adaptational changes at the beta-arrestin1 level, which in turn attenuates agonist-mediated desensitization and internalization of G-protein-coupled receptors.

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.

Morphine Metabolism in the Pregnant Guinea Pig and Her Pups

The study of chronic in utero exposure to heroin and morphine in the human is limited by polysubstance abuse. The guinea pig was used as a model for the human to determine the in vivo and in vitro effect of chronic morphine exposure on morphine metabolism in the pregnant dam and her offspring. In vivo pharmacokinetics of morphine were examined in pregnant guinea pigs following pretreatment with either saline or morphine. In vitro hepatic enzyme kinetics were also examined in a similar group of pregnant dams and their fetuses. Additional pregnant dams were allowed to give birth and their pups’ enzyme kinetics were studied at 1, 3, and 7 days. Apparent V_MAX for the formation of both morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation was significantly increased in the morphine-treated pregnant guinea pig. However, no effect of morphine treatment was detectable on the in vivo pharmacokinetics of morphine in the pregnant dam. The apparent morphine K_M for the formation of M3G was significantly different than the apparent K_M for the formation of M6G. Significant age effects on the enzyme kinetics were found. The apparent V_MAX for the formation of both glucuronides increased through the neonatal period. Through literature comparisons, the guinea pig was shown to have in vivo pharmacokinetics similar to the pregnant human, and the guinea pig pups were found to have enzyme development consistent with in vivo pharmacokinetic development seen in human neonates, infants and children.

Chronic morphine administration enhances the expression of Kv1.5 and Kv1.6 voltage-gated K+ channels in rat spinal cord.

Prolonged opiate administration leads to the development of tolerance and dependence. These phenomena are accompanied by selective regulation of distant cellular proteins and mRNAs, including ionic channels. Acute opiate administration differentially affects voltage-dependent K+ currents. Whereas, opiate activation of K+ channels is well established opioid-induced inhibition of K+ conductance has also been studied. In this study, we focused on the effect of chronic morphine exposure on voltage-dependent Shaker-related Kv1.5 and Kv1.6 K+ channel gene expression and on Kv1.5 protein levels in the rat spinal cord. Several experimental approaches including in-situ hybridization, RNAse protection, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were employed. We found that motor neurons are highly enriched in Kv1.5 and Kv1.6 mRNA and in Kv1.5 channel protein. Moreover, we found significant increases in the amount of mRNA encoding for these two K+ channels and in Kv1.5 channel protein in the spinal cord of morphine-treated rats, compared with controls. For example, quantitative in-situ hybridization, revealed a 2.1 +/- 0.15- and 2.3 +/- 0.5-fold increase in Kv1.5 and Kv1.6 channel mRNA levels, respectively. Similar results were obtained by semiquantitative RT-PCR analyses. Kv1.5 protein level was increased by 1.9-fold in the spinal cord or morphine-treated rats. Our results suggest that Kv1.5 and Kv1.6 Shaker K+ channels play an important role in regulating motor activity that increases in mRNA and protein levels of the spinal cord K+ channels after chronic morphine exposure could be viewed as a cellular adaptation which compensates for a persistent opioid-induced inhibition of K+ channel activity. These alterations may account, in part, for the cellular events leading to opiate tolerance and dependence.